PROJECT SUMMARY/ABSTRACT Osteomyelitis is an inflammatory state of bone most commonly triggered by the Gram-positive bacterium Staphylococcus aureus. Staphylococcal infection of bone is both common and highly morbid. During osteomyelitis, the structural matrix and vascular architecture of bone is destroyed, resulting in regions of necrotic, avascular tissue that increase risk of chronic infection. Extreme hypoxia occurs during osteomyelitis due to an altered vascular supply, inflammatory infiltration, and the metabolism of invasive S. aureus. The host responds to hypoxia by initiating hypoxia-inducible factor (HIF) signaling. HIF signaling, in turn, modulates bone remodeling. Beyond bone homeostasis and repair, HIF signaling is implicated in the regulation of antibacterial immunity. However, the role of hypoxia and HIF signaling during osteomyelitis is unknown and may serve as an important clinical target for optimizing immune responses to combat invasive infection. We have developed a powerful murine model of osteomyelitis that recapitulates the pathogenesis of human disease. Using this model, we demonstrated that extreme hypoxia is present at the infectious focus in a manner independent of non-infectious fracture healing. Our preliminary data demonstrate that VEGF, a well- characterized target of HIF signaling, is increased during osteomyelitis relative to mock-infected bone, consistent with the notion that HIF signaling is activated during invasive infection of bone. Under normoxia, we also observed that osteoblasts (bone-building cells) sense and respond to S. aureus stimulation in vitro by increasing the transcription of genes that regulate HIF signaling, control host-mediated bone remodeling, and mediate innate immune responses. The overarching hypothesis of this proposal is that tissue oxygenation and host hypoxic signaling responses influence antibacterial immunity and bone repair during S. aureus osteomyelitis. I will test this hypothesis with two integrated Specific Aims. In Specific Aim 1, I will define the role of oxygenation in mediating osteoblast innate immune responses and bone homeostasis in response to S. aureus stimulation in vitro. In Specific Aim 2, I will determine the impact of HIF signaling on antibacterial immunity and bone remodeling during S. aureus osteomyelitis. Within the experiments of Aim 2, I will use in vivo analyses that will harness both mouse genetics and biomaterial science to alter HIF signaling during osteomyelitis and investigate the impact of HIF on immune responses and bone repair. Together these studies will 1) define the role of oxygenation in dictating the innate immune and bone remodeling responses of osteoblasts, 2) define the impact of osteoblast HIF signaling on antibacterial immunity and bone repair during S. aureus osteomyelitis, and 3) test the potential of HIF-targeted therapies as an anti-infective adjuvant for invasive infection of bone. Collectively, this proposal will elucidate how oxygenation impacts skeletal cell biology and the innate immune responses to bacterial pathogens while providing ideal training and mentorship to develop my future career as an independent investigator and physician-scientist.